Project Summary/Abstract We have developed PSC-RED, a chemically-defined scalable method to differentiate induced pluripotent stem cells (iPSCs) into enucleated cultured Red Blood Cells (cRBCs) and we have demonstrated that we can generate cells expressing a GPI-anchored, truncated fragment of ADAMTS13 that is able to efficiently cleave its von Willebrand (VWF) cognate recognition site, while inserted in the cytoplasmic membrane. The main objective of this proposal is to test whether transfusion of a few mL of therapeutic GPI- ADAMTS13-cRBCs could replace plasma exchange as a treatment for Thrombotic Thrombocytopenic Purpura (TTP). In Aim 1, we propose to produce cRBCs engineered site-specifically at the AAVS1 safe- harbor site to express variant forms of GPI-ADAMTS13 that are resistant to the auto-antibodies responsible for idiopathic TTP. We will validate the resistance of these GPI-ADAMTS13-cRBCs to a panel of plasmas from untreated TTP patients, characterize their cellular properties, in vitro using a battery of tests, and in vivo using a murine xeno-transfusion models based on injection of clodronate liposomes (CloLip) and Cobra Venom Factor (CVF) that allows human RBCs to survive multiple days in the mouse circulation. In Aim 2, we will directly test whether GPI-ADAMTS13 red blood cells (RBCs) can be used to compensate ADAMTS13 loss of activity in a fully immuno-competent animal model. We have engineered a mouse that express GPI-ADAMTS13 specifically in RBCs from the rosa26 locus. We proposed to characterize these cells and to transfuse them in a model of TTP based on injection of large amounts of recombinant VWF into ADAMTS13KO mice. If successful, these key experiments will provide a proof-of-principle that transfusion of RBCs carrying a membrane-bound ADAMTS13 can be used as a treatment for TTP. We have shown that our protocol to produce cRBCs can be used to differentiate olive baboon iPSCs into enucleated cRBCs. In Aim 3, we propose to characterize olive baboon GPI-ADAMTS13-cRBCs in vitro, and to measure the half-life and the enzymatic activity of iPSC-derived GPI-ADAMTS13-cRBCs in vivo, in a large animal model. Engineered cRBCs are a highly promising avenue of translational research in the transfusion and the drug delivery fields. Accomplishing the proposed Aims will provide pre-clinical data for a novel treatment for congenital and idiopathic TTP. The proposed experiments will also validate a powerful platform to produce and test therapeutic iPSC-derived cRBCs which could have many other applications.